The present invention relates generally to the field of cell physiology, and more particularly, to apoptosis. More specifically, the present invention relates to methods for detecting the anti-apoptotic activity and function of viral polypeptides and their interactions with cellular polypeptides. The present invention further relates to viral polypeptides having anti-apoptotic activity and also the mechanism by which such viral polypeptides function to regulate or modulate apoptosis or cell death. Moreover, the present invention relates to compounds that regulate or modulate the anti-apoptotic activity of viral polypeptides, and expression of polynucleotides encoding such polypeptides. Such regulation or modulation of anti-apoptotic activity can lead to the restoration or induction of apoptosis in virally-infected cells and, consequently, inhibit or diminish viral replication.
The present invention relates to novel compounds that regulate or modulate apoptosis and/or anti-apoptotic activity, and uses and methods of screening for such compounds, e.g., 1) novel compounds having anti-apoptotic activity, such as anti-apoptotic polypeptides and the polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; and 2) novel compounds that inhibit or diminish the anti-poptotic activity of anti-apoptotic polypeptides and/or expression of polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; wherein the compounds include diagnostic and/or therapeutic compounds, and wherein the uses include diagnostic and/or therapeutic uses.
The polypeptides, and polynucleotides encoding such polypeptides, comprise viral polypeptides having anti-apoptotic activity, and/or polynucleotides encoding such polypeptides, respectively. Examples of such polypeptides are viral polypeptides of human cytomegalovirus (HCMV) having anti-apoptotic activity, such as pUL36, pUL37S, pUL37M, and pUL37L.
The compounds that regulate or modulate apoptosis and/or anti-apoptotic activity comprise polypeptide, polynucleotide (e.g., DNA and/or RNA), amino acid, nucleotide, and/or chemical compounds, including analogs and/or modified forms of such compounds, and/or synthetic and/or chemical compounds.
xe2x80x9cApoptosisxe2x80x9d refers to programmed cell death which occurs by an active, physiological process (Kerr, J. F., et al., 1972; Wyllie, A. H., 1980). Cells that die by apoptosis undergo characteristic morphological changes, including cell shrinkage and nuclear condensation and fragmentation. Apoptosis plays an important role in developmental processes, including morphogenesis, maturation of the immune system, and tissue homeostasis whereby cell numbers are limited in tissues that are continually renewed by cell division (Ellis, R. E., et aL, 1991; Oppenheim, R. W., et al., 1991; Cohen, J. J., et al., 1992; Raff, M. C. 1992). Moreover, apoptosis is an important cellular safeguard against tumorigenesis (Williams, G. T., 1991; Lane, D. P., 1993). Defects in the apoptotic pathway causing disregulated or aberrant apoptosis may contribute to the onset or progression of malignancies. Under certain conditions, cells undergo apoptosis in response to the forced expression of oncogenes, or other genes that drive cell proliferation (Askew, D., et al., 1991; Evan, G. I., et al., 1992; Rao, L., et al., 1992; Smeyne, R. J., et al., 1993).
A variety of diseases and degenerative disorders may involve aberrant or disregulated apoptosis, resulting in inappropriate or premature cell death or inappropriate cell proliferation (Barr, P. J., et al., 1994). For example, inhibition of cell death may contribute to disease in the immune system by allowing the persistence of self-reactive B and T cells, which consequently promotes autoimmune disease (Watanabe-Fukunaga et al., 1992). Moreover, cancer may result when cells that fail to die undergo further mutations leading to a transformed state of the cells (Korsmeyer, S. J., 1992).
The productive infection by certain viruses may depend on suppression of host cell death by anti-apoptotic viral gene products (Rao, L., et al., 1992; Ray, C. A., et al., 1992; White, E., et al., 1992; Vaux, D. L., et al., 1994), and inhibition of apoptosis can alter the course (i.e., lytic vs. latent) of viral infection (Levine, B., et al., 1993). Moreover, the widespread apoptosis of T lymphocytes triggered by HIV infection may, at least in part, be responsible for the immune system failure associated with AIDS (Gougeon, M., et al., 1993). The roles of apoptosis in normal and pathological cell cycle events are reviewed in Holbrook, N.J. et al., 1996. Importantly, apoptosis comprises an important antiviral defense mechanism in animals and humans by providing the means to rapidly eliminate virally infected cells and restrict viral propagation (O""Brien, 1998; Tschopp et al., 1998). Apoptosis of virally infected cells is triggered by killer cells of the immune system via Fas-ligand interaction with Fas and by granzyme-B-triggered caspase activation (Nagata and Golstein, 1995; Smyth and Trapani, 1998).
To counteract the host defense mechanism, many viruses encode genes that function to inhibit or diminish apoptosis in infected cells (O""Brien, 1998; Tschopp et al., 1998). This inhibition or diminution of apoptosis by viral gene products is achieved by a variety of mechanisms, including: 1) blocking and/or destruction of p53; 2) direct interaction with cellular polypeptides of apoptotic pathways, such as death-effector-domain-containing polypeptides [death-effector-domain motifs are defined in Hu et al., 1997], Bcl-2 family members, and caspases; or 3) by induction of cellular anti-apoptotic polypeptides (Pilder et al., 1984; Gooding et al., 1988; Clem et al., 1991; Hershberger et al., 1992; Brooks et al., 1995; Sedger and McFadden, 1996; Leopardi and Roizman, 1996; Leopardi et al., 1997; Razvi and Welsh, 1995; Teodoro and Branton, 1997; Vaux et al., 1994; Shen and Shenk, 1995; Duke et al., 1996; Vaux and Strasser, 1996; Thompson, 1995).
The prevalence and evolutionary conservation of anti-apoptotic viral genes suggests that suppression of apoptosis is a critical component of efficient viral propagation and/or persistence in vivo. In fact, some of the anti-apoptotic genes were found to be essential for the ability of the respective viruses to replicate and propagate. For example, mutants of human adenovirus that lack the expression of the E1B 19 kDa adenoviral analog of Bcl-2 induce massive apoptosis of infected cells (Teodoro and Branton, 1997) which, consequently, leads to reduced viral titers.
Human cytomegalovirus (HCMV) is widespread in human populations, and is of substantial clinical importance principally because of its pathogenicity in developing fetuses and immunocompromised individuals (Huang and Kowalik, 1993; Britt and Alford, 1996). In particular, those immunocompromised individuals undergoing organ and tissue transplants, or that have malignancies and are receiving immunosuppressive chemotherapy, or that have AIDS, are at greatest risk of HCMV-induced diseases. These diseases range from developmental abnormalities, mental retardation, deafness, mononucleosis, and chorioretinitis, to fatal diseases like interstitial pneumonitis and disseminated HCMV infections (Huang and Kowalik, 1993; Britt and Alford, 1996).
Human cytomegalovirus (HCMV) is a herpesvirus (Roizman, 1991). A number of herpesviruses were shown to induce an apoptotic host cell response, and to suppress this virus-induced apoptosis in the infected cells (Leopardi and Roizman, 1996; Leopardi et al., 1997; Bertin et al., 1997; Sieg et al., 1996). The genomes of several herpesviruses code for a variety of anti-apoptotic polypeptides such as: 1) Bcl-2 homologs, e.g., BHRF-1 of Epstein-Barr virus (Henderson et al., 1993), vbcl-2 of Kaposi""s sarcoma-associated herpesvirus (Sarid et al., 1997), and ORF16 of herpesvirus Saimiri (Nava et al., 1997); 2) a polypeptide that induces several cellular anti-apoptotic genes, e.g., LMP-1 of Epstein-Barr virus (Henderson et al., 1991; Wang et al., 1996; Fries et al., 1996); 3) a polypeptide interacting with FLICE (also called caspase 8), e.g., Equine herpesvirus type 2 polypeptide E8 (Bertin et al., 1997; Hu et al., 1997); and 4) two polypeptides with anti-apoptotic properties with a yet poorly characterized mechanism, ICP4 and US3 of HSV-1 (Leopardi and Roizman, 1996; Leopardi et al., 1997).
While there are several examples of anti-apoptotic genes encoded by other herpesviruses (Tschopp et al., 1998), little is known about the role of apoptosis in HCMV infections. It has been observed that HCMV-infected human cells acquire resistance towards apoptosis induced by serum withdrawal (Kovacs et al., 1996), and by infection of a mutant adenovirus which lacks the expression of the anti-apoptotic polypeptide E1B 19K (Zhu et al., 1995). Two immediate early polypeptides of HCMV, IE1 and IE2, were reported to each exhibit anti-apoptotic activity in some settings (Zhu et al., 1995). However, these viral polypeptides did not suppress apoptosis in other assays (see below).
The HCMV genome (AD169 strain) has been completely sequenced (Chee et al. 1990;
Mocarski, 1996). The 230 kb HCMV genome is predicted to encode over 200 polypeptides, many of which have undefined functions (Chee et al., 1990; Mocarski et al., 1996), and none of which bears overt homology to known classes of cell death suppressors (e.g., the Bcl-2 family). Whether most of the HCMV genes are expressed and have any functional importance for HCMV replication remains unknown. In fact, a number of the predicted ORFs were found to be dispensable for the replication and/or propagation of HCMV in cultured cells (Mocarski, 1996).
Aside from IE1 and IE2, no other HCMV anti-apoptotic genes have been identified, and no homology to any of the known anti-apoptotic polypeptides has been found in the HCMV genome. Prior to the discoveries embodied herein, little was known about the UL36 and UL37 genes of HCMV. On the basis of DNA sequence analysis and RNA transcription studies, it was predicted that UL36 has two exons which encode a polypeptide product pUL36, and that UL37 encodes two polypeptide products; pUL37S encoded by the first exon (also called pUL37x1, see Tenney and Colberg-Poley, 1991a), and pUL37L (also called gpUL37, see Zhang et al., 1996) encoded by all three exons (Chee et al., 1990; Tenney and Colberg-Poley, 1991a,b). The expression of pUL37L in HCMV-infected cells has been detected. However, prior to the discoveries embodied herein, it was not clear whether the hypothetical polypeptide pUL37S was expressed in HCMV-infected cells. Moreover, pUL37M had not yet been discovered.
Today, there are only very limited treatment options available for cytomegalovirus infections, and treatment is often associated with high toxicity and the generation of drug resistance (Hirsch, 1994; White and Fenner, 1994; Lalezari et al., 1997). Several potential drug targets for herpesviruses have recently been identified (White and Fenner, 1994, page 267, Table 16.1). Most of the antiviral compounds thus far developed, function to prevent or inhibit viral replication. For example, the currently available antiviral compounds, Gancyclovir, Foscamet (PFA, phosphonoformic acid) and Cidofovir all act as inhibitors of viral DNA polymerase. There are no available antiviral compounds that function to inhibit or diminish viral infection or to eliminate virally infected cells by regulating or modulating the anti-apoptotic activity of viral polypeptides and/or the expression of viral genes or polynucleotides encoding polypeptides having anti-apoptotic activity, and thereby inducing or restoring apoptosis in virally infected cells.
The present invention relates to the discovery of methods for detecting the anti-apoptotic function of viral polypeptides and their interactions with cellular polypeptides. The present invention further relates to the discovery of several viral polypeptides having anti-apoptotic activity and also the mechanism by which such viral polypeptides function to regulate or modulate apoptosis and cell death. Moreover, the present invention relates to the discovery of compounds that regulate or modulate the anti-apoptotic activity of viral polypeptides and expression of polynucleotides encoding such polypeptides. Such regulation or modulation of anti-apoptotic activity can lead to the restoration or induction of apoptosis in virally-infected cells and, consequently, inhibit or diminish viral replication.
Thus, these findings provide an approach for the rational design of drugs that regulate or modulate apoptosis and/or anti-apoptotic activity, e.g., by: 1) restoring or inducing anti-apoptotic activity, and thereby inhibiting or diminishing apoptosis; or, alternatively, 2) inhibiting or diminishing anti-apoptotic activity, and thereby restoring or inducing apoptosis.
Accordingly, an object of the present invention is: 1) to provide novel compounds having anti-apoptotic activity, such as anti-apoptotic polypeptides and the polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; and 2) to provide novel compounds that inhibit or diminish the anti-apoptotic activity of anti-apoptotic polypeptides and polynucleotides encoding such polypeptides, and in vitro and in vivo uses and methods of screening for such compounds; wherein the compounds include diagnostic and/or therapeutic compounds, and wherein the uses include diagnostic and/or therapeutic uses. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
An important embodiment of the present invention is the treatment of diseases, particularly diseases where apoptosis is disregulated or aberrant. The methods of treatment have the potential to change the natural progression of such diseases by regulating or modulating apoptosis and/or anti-apoptotic activity. Depending on the disease, the methods of treatment will: 1) restore or induce anti-apoptotic activity and thereby inhibit or diminish apoptosis; or 2) inhibit or diminish anti-apoptotic activity and thereby restore or induce apoptosis. The invention provides therapeutic compounds and methods of using these compounds to treat such diseases. Accordingly, depending on the disease to be treated, the therapeutic compounds comprise: 1) polypeptides having anti-apoptotic activity and/or polynucleotides encoding such polypeptides; and/or 2) compounds that inhibit or diminish the anti-apoptotic activity of such polypeptides and/or expression of polynucleotides encoding such polypeptides.
An embodiment of the present invention provides methods of screening for and identifying polypeptides having anti-apoptotic activity in cells; and methods of screening for polynucleotides encoding such polypeptides.
In particular, an embodiment of the present invention provides methods of screening for and identifying viral polypeptides such as human cytomegalovirus (HCMV) polypeptides having anti-apoptotic activity in cells; and methods of screening for and identifying polynucleotides encoding such viral polypeptides. Examples of HCMV polypeptides include, polypeptides encoded by UL36, e.g., pUL36 (and/or any unspliced and/or alternatively spliced variants of the polypeptides encoded by UL36), wherein the polynucleotide sequence of UL36 is defined by nucleotides 49,776-48,246 of the HCMV AD169 genome. As reported herein, it is now known that this sequence encodes an inactive form of pUL36. The sequencing of an earlier passage of this strain (AD169early) revealed that the active form of pUL36 possesses a cysteine residue at position 131, rather than the arginine identified at this position in pUL36 of AD169.
More particularly, an embodiment of the present invention provides methods of screening for and identifying HCMV polypeptides pUL36, pUL37S, pUL37M, and pUL37L, having anti-apoptotic activity in cells; and methods of screening for and identifying polynucleotides encoding such HCMV polypeptides.
Even more particularly, an embodiment of the present invention provides methods for detecting at least one of HCMV polypeptides pUL36, pUL37S, pUL37M, or pUL37L, using polyclonal and/or monoclonal antibodies that specifically bind to at least one of pUL36 (or any unspliced or alternatively spliced variants of the polypeptides encoded by UL36), pUL37S, pUL37M, or pUL37L.
Another embodiment ol the present invention provides methods for detecting the anti-apoptotic activity of polypeptides. In particular, an embodiment of the present invention provides methods of detecting the anti-apoptotic activity of viral polypeptides such as HCMV polypeptides.
Even more particularly, an embodiment of the present invention provides methods of detecting the anti-apoptotic activity of at least one of HCMV polypeptides pUL36, pUL37S pUL37M, or pUL37L.
Another embodiment of the present invention provides methods of identifying compounds that specifically interact with or bind to at least one of HCMV polypeptides pUL36, pUL37S, pUL37M, or pUL37L in an in vitro binding assay. Such compounds comprise polypeptide (e.g., monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides methods of screening for and identifying physiological molecules that specifically bind to at least one of pUL36, pUL37S, pUL37M, or pUL37L,in an in vitro binding assay. Such physiological molecules comprise polypeptide, polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds. Examples of such physiological molecules are FADD, caspase 3, Apaf-1, BCl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form.
Another embodiment of the present invention provides methods of screening for and identifying compounds that interfere with the specific interaction of a physiological molecule with at least one of pUL36, pUL37S, pUL37M, or pUL37L. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides methods of screening for and identifying compounds that inhibit or diminish the specific binding of a physiological molecule to at least one of pUL36, pUL37S, pUL37M, or pUL37L, in an in vitro binding assay. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds. The physiological molecule comprises polypeptide, polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds. Examples of such physiological molecules are FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form.
More particularly, an embodiment of the present invention provides methods of screening for and identifying compounds that inhibit or diminish the specific binding of a physiological molecule to at least one of pUL36, pUL37S, pUL37M, or pUL37L, in an in vitro binding assay, wherein at least one of pUL36, pUL37S, pUL37M, or pUL37L is immobilized.
Another embodiment of the present invention provides methods of screening for and identifying polypeptides that specifically bind to at least one of pUL36 (or any unspliced or alternatively spliced variants of the polypeptides encoded by UL36), pUL37S, pUL37M, or pUL37L, in a double transformation assay.
Another embodiment of the present invention provides methods of screening for compounds that inhibit or diminish the specific binding of a polypeptide (e.g., FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form) to at least one of pUL36, pUL37S, pUL37M, or pUL37L, In cells. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides methods of screening for compounds that inhibit or diminish the specific binding of a polypeptide (e.g., FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form) to at least one of pUL36, pUL37S, pUL37M, or pUL37L, in a double transformation assay.
Another embodiment of the present invention provides methods of screening for compounds that inhibit or diminish anti-apoptotic activity in cells. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides methods of screening for compounds that inhibit or diminish the anti-apoptotic activity of HCMV polypeptides, in cells.
More particularly, an embodiment of the present invention provides methods of screening for compounds that inhibit or diminish the anti-apoptotic activity of at least one of HCMV polypeptides pUL36, pUL37S, pUL37M, or pUL37L, in cells.
Another embodiment of the present invention provides methods of screening for compounds that restore, induce, or modulate apoptotic activity in cells. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides methods of screening for compounds that restore, induce, or modulate apoptosis in cells transformed with a polynucleotide encoding at least one polypeptide having anti-apoptotic activity or at least one fragment of such a HCMV polypeptide.
More particularly, an embodiment of the present invention provides methods of screening for compounds that restore, induce, or modulate apoptosis in cells transformed with a polynucleotide encoding at least one HCMV polypeptide having anti-apoptotic activity or encoding at least one fragment of such a HCMV polypeptide.
Even more particularly, an embodiment of the present invention provides methods of screening for compounds that restore, induce, or modulate apoptosis in cells transformed with a polynucleotide encoding at least one of HCMV polypeptides pUL36, pUL37S, pUL37M, or pUL37L, or at least one fragment of such a HCMV polypeptide.
Another embodiment of the present invention provides methods of screening for compounds that regulate or modulate apoptosis and/or anti-apoptotic activity in cells. Such compounds comprise polypeptide (e.g., non-functional forms of anti-apoptotic polypeptides, and monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds. Examples of such compounds include modified and/or unmodified DNA and/or RNA antisense oligonucleotides.
Another embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a polypeptide having anti-apoptotic activity, or a polynucleotide encoding such a polypeptide; and thereby inhibiting, diminishing, or modulating apoptotic activity, and/or restoring, inducing, or modulating anti-apoptotic activity. Examples of such polypeptides are viral polypeptides, such as HCMV polypeptides, having anti-apoptotic activity and fragments of such HCMV polypeptides.
More particularly, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or at least one fragment thereof, or a homologue of such HCMV polypeptide or fragment thereof, or a polynucleotide encoding at least one of such HCMV polypeptides or encoding at least one fragment thereof, or a homologue of such HCMV polypeptides or fragment thereof, and thereby inhibiting, diminishing, or modulating apoptotic activity, and/or restoring, inducing, or modulating anti-apoptotic activity.
More particularly, an embodiment of the present invention provides methods of treating cells, that are target cells in a patient, by contacting the cells ex vivo with an effective amount of at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or at least one fragment thereof, or a homologue of such HCMV polypeptide or fragment thereof, or polynucleotide encoding at least one of such HCMV polypeptides or encoding at least one fragment thereof, or a homologue of such HCMV polypeptides or fragment thereof; and thereby inhibiting, diminishing, or modulating apoptotic activity, and/or restoring, inducing, or modulating anti-apoptotic activity. The ex vivo contact of the cells is by, for example, pressure-mediated delivery, gene gun delivery, and/or liposome delivery.
Even more particularly, an embodiment of the present invention provides methods of 5 treating cells, that are target cells in a patient, by contacting the cells ex vivo with an effective amount of a polynucleotide encoding at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or PUL37L, or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptide or fragment thereof, wherein the ex vivo contact comprises the transduction of the target cells with a synthetic and/or viral vector carrying the polynucleotide; and thereby inhibiting, diminishing, or modulating apoptotic activity, and/or restoring, inducing, or modulating anti-apoptotic activity. Examples of such synthetic and/or viral vectors for transducing the target cells such as retroviruses, adenovirus, adeno-associated virus, vaccinia virus, herpes simplex virus, avipox virus, and baculovirus.
Another embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a compound that specifically binds to a polypeptide having anti-apoptotic activity or that binds to a polynucleotide encoding such a polypeptide; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity. Such compounds comprise polypeptide (e.g., monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds. Examples of such compounds are synthetic peptides; modified or unmodified DNA or RNA antisense oligonucleotides; and other compounds that selectively bind to viral polypeptides and.not to cellular polypeptides. Moreover, the contact of cells is by, for example, pressure-mediated delivery, gene gun delivery, liposome delivery, and/or ex vivo contact.
In particular, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a compound that binds to at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or at least one fragment thereof, or a homologue of such HCMV polypeptide or fragment thereof, or a polynucleotide encoding at least one of such HCMV polypeptides or encoding at least one fragment thereof, or a homologue of such HCMV polypeptides or fragment thereof; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
In particular, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of an antisense-oligonucleotide that binds to a polynucleotide encoding at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptides or fragment thereof; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
More particularly, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a synthetic anti-sense oligonucleotide that specifically blocks the expression of a polynucleotide encoding a polypeptide having anti-apoptotic activity; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
Even more particularly, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a synthetic anti-sense oligonucleotide having a phosphorothioate backbone that specifically blocks the expression of a polynucleotide encoding at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptides or fragment thereof; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
Also, in particular, an embodiment of the present invention provides methods of treating cells having disregulated or aberrant apoptotic activity (prior to treatment), by contacting the cells with an effective amount of a polypeptide having anti-apoptotic activity, and/or a polynucleotide encoding such a polypeptide; and thereby inhibiting, diminishing, or modulating apoptotic activity, and/or restoring, inducing, or modulating anti-apoptotic activity. Such disregulated or aberrant apoptotic activity may be caused by a degenerative disorder characterized by inappropriate cell death or inappropriate cell proliferation. In particular, the disregulated or aberrant anti-apoptotic activity may be caused by a cancer, immune disorder, autoimmune disease, infectious disease, viral infection, or myocardial infarction or neuronal infarction in cardiovascular disease.
Also, in particular, an embodiment of the present invention provides methods of treating cells having (prior to contact) apoptotic activity potentiated or mediated by a death receptor such as Fas receptor (Fas) or Tumor Necrosis Factor Receptor 1 (TNF-R1), by contacting the cells with an effective amount of a compound that specifically binds to a polypeptide having anti-apoptotic activity or specifically binds to a polynucleotide encoding such a polypeptide; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
Also, in particular, an embodiment of the present invention provides methods of treating cells by contacting the cells with an effective amount of a compound that specifically binds to a polypeptide having anti-apoptotic activity or specifically binds to a polynuclcotide encoding such a polypeptide, wherein said polypeptide inhibits the activation of caspase 9, inhibits the activation of caspase 8, and/or inhibits the release of cytochrome c from mitochondria; and thereby inhibiting, diminishing, or modulating anti-apoptotic activity, and/or inducing, restoring, or modulating apoptotic activity.
Another embodiment of the present invention provides methods of enhancing the stability, growth, and/or productivity of cells by introducing into cells, by, for example, transfection or retrovirus infection, a polynucleotide encoding a polypeptide having anti-apoptotic activity, and expressing the polypeptide in the cells; wherein the cells under normal conditions do not express such a polypeptide. Examples of such cells are hybridomas, Chinese Hamster Ovary (CHO) cells, fibroblasts, lymphoid cells, haematopoietic cells, cells derived from the embryonic central nervous system, and cells derived from normal, dysplastic, or neoplastic tissue.
In particular, an embodiment of the present invention provides methods of enhancing the stability, growth, and/or productivity of cells by introducing into cells, by transfection or retrovirus infection, a polynucleotide encoding at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptides or fragment thereof, and expressing the polypeptide in the cells; wherein the cells under normal conditions do not express such a polypeptide.
Another embodiment of the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a polypeptide having anti-apoptotic activity, or a therapeutically effective amount of a polynucleotide encoding such a polypeptide.
In particular, an embodiment of the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or at least one fragment thereof, or a homologue of such HCMV polypeptides or fragment thereof; or a polynucleotide encoding at least one of such HCMV polypeptides or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptides or fragment thereof. In a preferred embodiment, the fragment of pUL37S comprises amino acid residues 5-34 and 118-147 of the native pUL37S protein. In another preferred embodiment, the homologue of pUL37S comprises amino acid residues 5-34 and 118-147 of the native pUL37S protein.
Another embodiment of the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound that specifically binds to a polypeptide having anti-apoptotic activity, or that specifically binds to a polynucleotide encoding such a polypeptide. Such compounds comprise polypeptide (e.g., monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise natural, semi-synthetic, and/or synthetic chemical compounds. An example of such a compound is a synthetic polypeptide that binds to the polypeptide having anti-apoptotic activity, or that binds to a polynucleotide encoding a polypeptide having anti-apoptotic activity. Another example of such a compound is an antisense oligonucleotide having a sequence complimentary to the sequence of the polynucleotide encoding a polypeptide having anti-apoptotic activity, wherein the antisense oligonucleotide may or may not comprise a phosphorothioate backbone.
In particular, an embodiment of the present invention provides pharmaceutical compositions comprising a therapeutically effective amount of a compound that specifically binds to at least one HCMV polypeptide pUL36, pUL37S, pUL37M, or pUL37L, or binds to at least one fragment thereof, or a homologue of such HCMV polypeptides or fragment thereof; or binds to a polynucleotide encoding at least one of such HCMV polypeptides or encoding at least one fragment thereof, or encoding a homologue of such HCMV polypeptides or fragment thereof.
Another embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in a cell, wherein the polypeptide comprises a viral polypeptide.
In particular, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises a human cytomegalovirus (HCMV) polypeptide other than pUL37S and UL37L.
More particularly, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises the amino acid sequence of pUL36 or pUL37M.
Also more particularly, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises a fragment of the amino acid sequence of pUL36, pUL37S, pUL37M or pUL37L. In a preferred embodiment, the fragment of pUL37S comprises amino acid residues 5-34 and 118-147 of the native pUL37S protein. Again more particularly, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises a sequence homologous to the amino acid sequence of pUL36, pUL37S, pUL37M, or pUL37L. In another preferred embodiment, the homologue of pUL37S comprises amino acid residues 5-34 and 118-147 of the native pUL37S protein.
Also, more particularly, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises a polypeptide encoded by UL36 (or any unspliced or alternatively spliced variants of the polypeptide encoded by UL36); and wherein the nucleotide sequence of UL36 is defined by nucleotides 49,776-48,246 of the HCMV AD169 genome.
In particular, an embodiment of the present invention provides an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide is encoded by nucleotides 49,776-48,246 of the HCMV AD169 genome and is pUL36.
Another embodiment of the present invention provides a compound that specifically binds to an isolated or synthetic polypeptide having anti-apoptotic activity in a cell, wherein the polypeptide comprises a viral polypeptide, and wherein the compound inhibits, diminishes, or modulates anti-apoptotic activity and/or restores, induces, or modulates apoptosis. Such compounds comprise polypeptide (e.g., monoclonal and/or polyclonal antibodies), polynucleotide (e.g., DNA and/or RNA), amino acid, and/or nucleotide compounds, including analogs and/or modified forms of such compounds. Additionally, such compounds comprise atural, semi-synthetic, and/or synthetic chemical compounds.
In particular, an embodiment of the present invention provides a compound that pecifically binds to an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide comprises a human cytomegalovirus (HCMV) polypeptide, and wherein the compound inhibits, diminishes, or modulates anti-apoptotic activity, and/or restores, induces, or modulates apoptosis. Examples of such compounds are monoclonal and/or polyclonal antibody that bind to at least one of pUL36, pUL37S, pUL37M, or pUL37L. More particularly, an embodiment of the present invention provides a compound that specifically binds to a physiological molecule that is bound by a polypeptide having anti-apoptotic activity in cells, wherein the physiological molecule is FADD, caspase 3, Apaf-1, BCl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form, and the polypeptide comprises the amino acid sequence of pUL36, pUL37S, pUL37M or pUL37L, or a fragment thereof, or a homologue of the polypeptide or fragment thereof, wherein the compound inhibits, diminishes, or modulates anti-apoptotic activity, and/or restores, induces, or modulates apoptosis
Also, more particularly, an embodiment of the present invention provides a compound that specifically binds to an isolated or synthetic polypeptide having anti-apoptotic activity in cells wherein the polypeptide comprises a polypeptide encoded by UL36 (or any unspliced or alternatively spliced variants of the polypeptide encoded by UL36); wherein the nucleotide sequence of UL36 is defined by nucleotides 49,776-48,246 of the HCMV AD169 genome; and wherein the compound inhibits, diminishes, or modulates anti-apoptotic activity, and/or restores, induces, or modulates apoptosis.
In particular, an embodiment of the present invention provides a compound that binds specifically to an isolated or synthetic polypeptide having anti-apoptotic activity in cells, wherein the polypeptide is encoded by nucleotides 49,776-48,246 of the HCMV AD169 genome and is pUL36, and wherein the compound inhibits, diminishes, or modulates anti-apoptotic activity, and/or restores, induces, or modulates apoptosis.
Another cmbodiment of the present invention provides a first isolated or synthetic polypeptide that specifically binds to a second isolated or synthetic polypeptide having anti-apoptotic activity in cells, and thereby the first polypeptide prevents the second polypeptide from binding to FADD, caspase 3, Apaf-1, BCl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form. Examples of the second polypeptide are HCMV polypeptides having anti-apoptotic activity, such as pUL36, pUL37S, pUL37M, and pUL37L.
In particular, an embodiment of the present invention provides a first isolated or synthetic polypeptide that specifically binds to a second isolated or synthetic polypeptide having anti-apoptotic activity in cells, and thereby the first polypeptide prevents the second polypeptide from binding to FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form, wherein the second polypeptide comprises a sequence homologous to the amino acid sequence pUL36, pUL37S, pUL37M or pUL37L.
In particular, an embodiment of the present invention provides a first isolated or synthetic polypeptide that specifically binds to a second isolated or synthetic polypeptide having anti-apoptotic activity in cells, and thereby the first polypeptide prevents the second polypeptide from binding to FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form, wherein the second polypeptide comprises a polypeptide encoded by UL36 (or any unspliced or alternatively spliced variants of the polypeptide encoded by UL36); and wherein the nucleotide sequence of UL36 is defined by nucleotides 49,776-48,246 of the HCMV AD1169 genome.
Also, in particular, an embodiment of the present invention provides a first isolated or synthetic polypeptide that binds specifically to a second isolated or synthetic polypeptide having anti-apoptotic activity in cells, and thereby the first polypeptide prevents the second polypeptide from binding to FADD, caspase 3, Apaf-1, Bcl-xL, Bcl-2, Bak, ICE, Bax, BNIP-3, ANT and caspase 8 in its pro- or activated form, and wherein the second polypeptide is encoded by nucleotides 49,776-48,246 of the HCMV AD169 genome and is pUL36.